Protein ubiquitination is a fundamental biological process that regulates all aspects of human biology. Covalent modification of proteins with polyubiquitin chains tags them for proteasomal degradation, while monoubiquitination regulates endocytosis, signal transduction, nuclear import, and gene transcription. To this end, ubiquitin ligase enzymes (~600 known) represent a vast and completely unexplored area of druggable genome. The complexity and diversity of ubiquitin ligases rivals that of three other large families of enzymes: kinases (~500), proteases (~600), and GPCRs (~800). There are 25 FDA approved kinase inhibitor drugs, ~30 FDA approved protease inhibitor drugs, and about 30% of all medications target GPCRs. In stark contrast, there are only 3 FDA approved drugs that target ubiquitin ligases: teratogenic compounds thalidomide, and its analogues lenalidomide and pomalidomide. Significantly, germline and somatic mutations of E3 ligase genes are linked to many human diseases such as cancers, autoimmune diseases, neurodegenerative diseases, and hypertensive disorders. Therefore E3 ligases have recently emerged as prominent targets for basic research and drug discovery. In this proposal, we focus our efforts on developing small molecule inhibitors of HECT E3 ligase Nedd4-1, which is an essential gene in mice and is a positive regulator of IGF-1 and insulin cell growth pathways, pathways that increase the risk and promote the growth of human cancers. Specific aims are as follows (1) To discover covalent inhibitors of Nedd4-1. We used an irreversible tethering technology, to identify two first-in-class covalent inhibitors of Nedd4-1 enzyme, and visualized their binding mode using X-ray crystallography. We propose to investigate biochemical mechanism of action of identified Nedd4-1 inhibitors, (2) conduct structure-activity relationship (SAR) studies of identified Nedd4 inhibitors, (3) to conduct cell based studies of Nedd4-1 inhibitors to show that they inhibit IGF-1/Insulin cell growth pathways in cells. In summary, the proposed research program outlines new concepts to discover covalent small molecule inhibitors of HECT E3 ubiquitin ligase Nedd4-1. The proposed research program will serve as a foundation to initiate novel research directions to discover and optimize small molecule probes targeting other HECT E3 ligases (28 known).